Many cytokines have shown potent antitumor activities in preclinical experiments and represent promising agents for cancer therapy. However, despite encouraging results in animal models, only a few cytokines, such as Proleukin 1 (IL2), Roferon A1 (interferon alpha-2a [IFNα 2a]), Intron A1 (IFNα 2b), Beromun 1 (recombinant TNFα) are approved as anticancer drugs. Current indications for cytokines include metastatic renal cell cancer, malignant melanoma, hairy cell leukemia, chronic myeloid lymphoma, sarcoma and multiple myeloma. The cytokines may be either administered alone or in combination with chemotherapy.
A further difficulty with pro-inflammatory cytokines in particular is that their use in therapy is often hindered by substantial toxicity even at low doses, which prevents the escalation to therapeutically active doses (Hemmerle et al. (2013) Br. J. Cancer 109, 1206-1213).
In an attempt to increase the therapeutic index of certain cytokines, antibody-cytokine fusion proteins (also referred to as “immunocytokines”) have been proposed. In these conjugates, the antibody serves as a “vehicle” for a selective accumulation at the site of disease, while the cytokine payload is responsible for the therapeutic activity (Pasche & Neri, 2012, Drug Discov. Today, 17, 583). Certain immunocytokines based on pro-inflammatory payloads (such as IL2, IL4, IL12, and TNFα) display potent anti-cancer activity in mouse models (Hess et al., 2014, Med. Chem. Comm., 5, 408) and have produced encouraging results in patients with both solid tumours and hematological malignancies (Eigentler et al., 2011, Clin. Cancer Res. 17, 7732-7742; Papadia et al., 2013, J. Surg. Oncol. 107, 173-179; Gutbrodt et al., 2013, Sci. Transl. Med. 5, 201-204; Weide et al., 2014, Cancer Immunol. Res. 2, 668-678; Danielli et al., 2015, Cancer Immunol. Immunother. 64, 113-121]. The F8 antibody (specific to the alternatively-spliced EDA domain of fibronectin, a marker of tumor angiogenesis; Rybak et al. (2007) Cancer Res. 67, 10948-10957) has been used for tumor targeting, both alone and fused to either TNF or IL2 (Villa et al. (2008) Int. J. Cancer 122, 2405-2413; Hemmerle et al. (2013) Br. J. Cancer 109, 1206-1213; Frey et al. (2008) J. Urol. 184, 2540-2548).
In some cases, immunocytokines can mediate tumor eradication in mouse models of cancer when used as single agents (Gutbrodt et al., 2013, Sci. Transl. Med. 5, 201-204]. In most cases, however, a single immunocytokine product is not able to induce complete cancer eradication. However, cancer cures have been reported for combinations of immunocytokines with cytotoxic agents (Moschetta et al., 2012, Cancer Res. 72, 1814-1824], intact antibodies (Schliemann et al., 2009, Blood, 113, 2275-2283] and external beam radiation (Zegers et al., 2015, Clin. Cancer Res., 21, 1151-1160).
In addition, several combinations of immunocytokines have been used in therapy. For example, conjugates L19-IL2 and L19-TNFα were able to cure neuroblastoma in a fully syngeneic mouse model of the disease, whereas the individual immunocytokines used as single agents did not result in eradication of the disease (Balza et al., 2010, Int. J. Cancer, 127, 101). The combination of IL2 and TNFα payloads has also shown promising results in clinical trials. The fusion proteins L19-IL2 and L19-TNF were shown to potently synergize for the intralesional treatment of certain solid tumors in the mouse (Schwager et al., 2013, J. Invest. Dermatol. 133, 751-758). The corresponding fully human fusion proteins have been administered intralesionally to patients with Stage IIIC melanoma (Danielli et al., 2015, Cancer Immunol. Immunother. 64, 113-121), showing better results compared to the intralesional administration of interleukin-2 (Weide et al., 2011, Cancer—116, 4139-4146) or of L19-IL2 (Weide et al., 2014, Cancer Immunol. Immunother. 2, 668-678). However, the genetic fusion of a cytokine to an antibody does not always result in increased efficacy. For example, the fusion of Interleukin-17 to a targeting antibody did not reduce tumour growth (Pasche et al., 2012, Angiogenesis 15, 165-169).
There have also been attempts to generate “dual immunocytokines” in which an antibody is genetically fused to two different cytokines. For instance interleukin-12 (IL12) and TNFα have been incorporated into a single molecular entity. However, these attempts have not been successful and have not led to clinical development programs.
Specifically, a triple fusion, consisting of: (i) the L19 antibody in scFv format (specific to the alternatively-spliced EDB domain of fibronectin, a marker of tumor angiogenesis); (ii) murine TNFα; and (iii) murine IL12 in single-chain format has been described (Halin et al., 2003, Cancer Res., 63, 3202-3210). The fusion protein could be expressed and purified to homogeneity. The fusion protein also bound to the cognate antigen with high affinity and specificity, but (unlike L19-TNFα and L19-IL12) failed to localize to solid tumors in vivo, as evidenced by quantitative biodistribution studies in tumor-bearing mice.
Bi-functional cytokine fusion proteins in which the cytokines were linked to an intact antibody (or the Fc portion of an antibody) have also been described. These fusion proteins comprised interleukin-2/interleukin-12 (IL-2/IL-12), or interleukin-4/granulocyte-macrophage colony-stimulating factor (IL-4/GM-CSF). Cytokine activity was retained in constructs where the cytokines were fused in tandem at the carboxyl terminus of the Fc or antibody heavy (H) chain, as well as in constructs where one cytokine was fused at the carboxyl terminus of the H chain while the second cytokine was fused to the amino terminus of either the H or light (L) chain variable region. Antigen binding of the antibody-cytokine fusion proteins was maintained. However, therapeutic activities in vivo were reported only for gene therapy applications (i.e., tumor cells transfected with the appropriate IL2/IL12 immunocytokines), but not with therapeutic proteins (Gillies et al., 2002, Cancer Immunol. Immunother., 51, 449).
As a result of the intrinsic complexity of successfully expressing immunoconjugates comprising two cytokines in a single molecule (also referred to as “dual immunocytokines”), as well as the unpromising results obtained with such molecules as discussed above, these molecular formats have not been pursued for clinical applications.